METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA

ABSTRACT

The. invention relates to a method for inducing or promoting skipping of exon 45 of DMD pre-mRNA in a Duchenne Muscular Dystrophy patient, preferably in an isolated (muscle) cell, the method comprising providing said cell with an antisense molecule that binds to a continuous stretch of at least 21 nucleotides within said exon. The invention further relates to such antisense molecule used in said method.

FIELD

The invention relates to the field of genetics, more specifically humangenetics. The invention in particular relates to the modulation ofsplicing of the human Duchenne Muscular Dystrophy pre-mRNA.

BACKGROUND OF THE INVENTION

Myopathies are disorders that result in functional impairment ofmuscles. Muscular dystrophy (MD) refers to genetic diseases that arecharacterized by progressive weakness and degeneration of skeletalmuscles. Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy(BMD) are the most common childhood forms of muscular dystrophy. Theyare recessive disorders and because the gene responsible for DMD and BMDresides on the X-chromosome, mutations mainly affect males with anincidence of about 1 in 3500 boys.

DMD and BMD are caused by genetic defects in the DMD gene encodingdystrophin, a muscle protein that is required for interactions betweenthe cytoskeleton and the extracellular matrix to maintain muscle fiberstability during contraction. DMD is a severe, lethal neuromusculardisorder resulting in a dependency on wheelchair support before the ageof 12 and DMD patients often die before the age of thirty due torespiratory- or heart failure. In contrast, BMD patients often remainambulatory until later in life, and have near normal life expectancies.DMD mutations in the DMD gene are mainly characterized by frame shiftinginsertions or deletions or nonsense point mutations, resulting in theabsence of functional dystrophin. BMD mutations in general keep thereading frame intact, allowing synthesis of a partly functionaldystrophin.

During the last decade, specific modification of splicing in order torestore the disrupted reading frame of the DMD transcript has emerged asa promising therapy for Duchenne muscular dystrophy (DMD) (van Ommen,van Deutekom, Aartsma-Rus, Curr Opin Mol Ther. 2008; 10(2):140-9,Yokota, Duddy, Partidge, Acta Myol. 2007; 26(3):179-84, van Deutekom etal., N Engl J Med. 2007; 357(26):2677-86).

Using antisense oligonucleotides (AONs) interfering with splicingsignals the skipping of specific exons can be induced in the DMDpre-mRNA, thus restoring the open reading frame and converting thesevere DMD into a milder BMD phenotype (van Deutekom et al. Hum MolGenet. 2001; 10: 1547-54; Aartsma-Rus et al., Hum Mol Genet 2003;12(8):907-14.). In vivo proof-of-concept was first obtained in the mdxmouse model, which is dystrophin-deficient due to a nonsense mutation inexon 23.

Intramuscular and intravenous injections of AONs targeting the mutatedexon 23 restored dystrophin expression for at least three months (Lu etal. Nat Med. 2003; 8: 1009-14; Lu et al., Proc Natl Acad Sci USA. 2005;102(1):198-203). This was accompanied by restoration ofdystrophin-associated proteins at the fiber membrane as well asfunctional improvement of the treated muscle. In vivo skipping of humanexons has also been achieved in the hDMD mouse model, which contains acomplete copy of the human DMD gene integrated in chromosome 5 of themouse (Bremmer-Bout et al. Molecular Therapy. 2004; 10: 232-40; 't Hoenet al. J Biol Chem. 2008; 283: 5899-907).

As the majority of DMD patients have deletions that cluster in hotspotregions, the skipping of a small number of exons is applicable torelatively large numbers of patients. The actual applicability of exonskipping can be determined for deletions, duplications and pointmutations reported in DMD mutation databases such as the Leiden DMDmutation database available at www.dmd.nl. Therapeutic skipping of exon45 of the DMD pre-mRNA would restore the open reading frame of DMDpatients having deletions including but not limited to exons 12-44,18-44, 44, 46, 46-47, 46-48, 46-49, 46-51, 46-53, 46-55, 46-59, 46-60 ofthe DMD pre-mRNA, occurring in a total of 16% of all DMD patients with adeletion (Aartsma-Rus and van Deutekom, 2007, Antisense Elements(Genetics) Research Focus, 2007 Nova Science Publishers, Inc).Furthermore, for some DMD patients the simultaneous skipping of one ofmore exons in addition to exon 45, such as exons 51 or 53 is required torestore the correct reading frame. None-limiting examples includepatients with a deletion of exons 46-50 requiring the co-skipping ofexons 45 and 51, or with a deletion of exons 46-52 requiring theco-skipping of exons 45 and 53.

Recently, a first-in-man study was successfully completed where an AONinducing the skipping of exon 51 was injected into a small area of thetibialis anterior muscle of four DMD patients. Novel dystrophinexpression was observed in the majority of muscle fibers in all fourpatients treated, and the AON was safe and well tolerated (van Deutekomet al. N Engl J Med. 2007; 357: 2677-86).

Most AONs studied contain up to 20 nucleotides, and it has been arguedthat this relatively short size improves the tissue distribution and/orcell penetration of an AON. However, such short AONs will result in alimited specificity due to an increased risk for the presence ofidentical sequences elsewhere in the genome, and a limited targetbinding or target affinity due to a low free energy of the AON-targetcomplex. Therefore the inventors decided to design new and optionallyimproved oligonucleotides that would not exhibit all of these drawbacks.

DESCRIPTION OF THE INVENTION

Method

In a first aspect, the invention provides a method for inducing and/orpromoting skipping of exon 45 of DMD pre-mRNA in a patient, preferablyin an isolated cell of said patient, the method comprising providingsaid cell and/or said patient with a molecule that binds to a continuousstretch of at least 21 nucleotides within said exon.

Accordingly, a method is herewith provided for inducing and/or promotingskipping of exon 45 of DMD pre-mRNA, preferably in an isolated cell of apatient, the method comprising providing said cell and/or said patientwith a molecule that binds to a continuous stretch of at least 21nucleotides within said exon.

It is to be understood that said method encompasses an in vitro, in vivoor ex vivo method.

As defined herein a DMD pre-mRNA preferably means the pre-mRNA of a DMDgene of a DMD or BMD patient. The DMD gene or protein corresponds to thedystrophin gene or protein.

A patient is preferably intended to mean a patient having DMD or BMD aslater defined herein or a patient susceptible to develop DMD or BMD dueto his or her genetic background.

Exon skipping refers to the induction in a cell of a mature mRNA thatdoes not contain a particular exon that is normally present therein.Exon skipping is achieved by providing a cell expressing the pre-mRNA ofsaid mRNA with a molecule capable of interfering with sequences such as,for example, the splice donor or splice acceptor sequence that are bothrequired for allowing the enzymatic process of splicing, or a moleculethat is capable of interfering with an exon inclusion signal requiredfor recognition of a stretch of nucleotides as an exon to be included inthe mRNA. The term pre-mRNA refers to a non-processed or partlyprocessed precursor mRNA that is synthesized from a DNA template in thecell nucleus by transcription.

Within the context of the invention inducing and/or promoting skippingof an exon as indicated herein means that at least 1%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or more of the DMD mRNA in one or more(muscle) cells of a treated patient will not contain said exon. This ispreferably assessed by PCR as described in the examples.

Preferably, a method of the invention by inducing or promoting skippingof exon 45 of the DMD pre-mRNA in one or more cells of a patientprovides said patient with a functional dystrophin protein and/ordecreases the production of an aberrant dystrophin protein in saidpatient. Therefore a preferred method is a method, wherein a patient ora cell of said patient is provided with a functional dystrophin proteinand/or wherein the production of an aberrant dystrophin protein in saidpatient or in a cell of said patient is decreased Decreasing theproduction of an aberrant dystrophin may be assessed at the mRNA leveland preferably means that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%,10%, 5% or less of the initial amount of aberrant dystrophin mRNA, isstill detectable by RT PCR. An aberrant dystrophin mRNA or protein isalso referred to herein as a non-functional dystrophin mRNA or protein.A non functional dystrophin protein is preferably a dystrophin proteinwhich is not able to bind actin and/or members of the DGC proteincomplex. A non-functional dystrophin protein or dystrophin mRNA doestypically not have, or does not encode a dystrophin protein with anintact C-terminus of the protein.

Increasing the production of a functional dystrophin in said patient orin a cell of said patient may be assessed at the mRNA level (by RT-PCRanalysis) and preferably means that a detectable amount of a functionaldystrophin mRNA is detectable by RT PCR. In another embodiment, 1%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectabledystrophin mRNA is a functional dystrophin mRNA. Increasing theproduction of a functional dystrophin in said patient or in a cell ofsaid patient may be assessed at the protein level (by immunofluorescenceand western blot analyses) and preferably means that a detectable amountof a functional dystrophin protein is detectable by immunofluorescenceor western blot analysis. In another embodiment, 1%, 5%, 10%, 20%/a,30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophinprotein is a functional dystrophin protein.

As defined herein, a functional dystrophin is preferably a wild typedystrophin corresponding to a protein having the amino acid sequence asidentified in SEQ ID NO: 1. A functional dystrophin is preferably adystrophin, which has an actin binding domain in its N terminal part(first 240 amino acids at the N terminus), a cystein-rich domain (aminoacid 3361 till 3685) and a C terminal domain (last 325 amino acids atthe C terminus) each of these domains being present in a wild typedystrophin as known to the skilled person. The amino acids indicatedherein correspond to amino acids of the wild type dystrophin beingrepresented by SEQ ID NO: 1. In other words, a functional dystrophin isa dystrophin which exhibits at least to some extent an activity of awild type dystrophin. “At least to some extent” preferably means atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of acorresponding activity of a wild type functional dystrophin. In thiscontext, an activity of a functional dystrophin is preferably binding toactin and to the dystrophin-associated glycoprotein complex (DGC)(Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne MuscularDystrophy mutation database: an overview of mutation types andparadoxical cases that confirm the reading-frame rule, Muscle Nerve, 34:135-144). Binding of dystrophin to actin and to the DGC complex may bevisualized by either co-immunoprecipitation using total protein extractsor immunofluorescence analysis of cross-sections, from a muscle biopsy,as known to the skilled person.

Individuals or patients suffering from Duchenne muscular dystrophytypically have a mutation in the DMD gene that prevent synthesis of thecomplete dystrophin protein, i.e of a premature stop prevents thesynthesis of the C-terminus. In Becker muscular dystrophy the DMD genealso comprises a mutation compared to the wild type gene but themutation does typically not induce a premature stop and the C-terminusis typically synthesized. As a result a functional dystrophin protein issynthesized that has at least the same activity in kind as the wild typeprotein, not although not necessarily the same amount of activity. Thegenome of a BMD individual typically encodes a dystrophin proteincomprising the N terminal part (first 240 amino acids at the Nterminus), a cystein-rich domain (amino acid 3361 till 3685) and a Cterminal domain (last 325 amino acids at the C terminus) but its centralrod shaped domain may be shorter than the one of a wild type dystrophin(Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne MuscularDystrophy mutation database: an overview of mutation types andparadoxical cases that confirm the reading-frame rule, Muscle Nerve, 34:135-144). Exon-skipping for the treatment of DMD is typically directedto overcome a premature stop in the pre-mRNA by skipping an exon in therod-shaped domain to correct the reading frame and allow synthesis ofthe remainder of the dystrophin protein including the C-terminus, albeitthat the protein is somewhat smaller as a result of a smaller roddomain. In a preferred embodiment, an individual having DMD and beingtreated by a method as defined herein will be provided a dystrophinwhich exhibits at least to some extent an activity of a wild typedystrophin. More preferably, if said individual is a Duchenne patient oris suspected to be a Duchenne patient, a functional dystrophin is adystrophin of an individual having BMD: typically said dystrophin isable to interact with both actin and the DGC, but its central rod shapeddomain may be shorter than the one of a wild type dystrophin(Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne MuscularDystrophy mutation database: an overview of mutation types andparadoxical cases that confirm the reading-frame rule, Muscle Nerve, 34:135-144). The central rod-shaped domain of wild type dystrophincomprises 24 spectrin-like repeats (Aartsma-Rus A et al, (2006), Entriesin the leiden Duchenne Muscular Dystrophy mutation database: an overviewof mutation types and paradoxical cases that confirm the reading-framerule, Muscle Nerve, 34: 135-144). For example, a central rod-shapeddomain of a dystrophin as provided herein may comprise 5 to 23, 10 to 22or 12 to 18 spectrin-like repeats as long as it can bind to actin and toDGC.

A method of the invention may alleviate one or more characteristics of amuscle cell from a DMD patient comprising deletions including but notlimited to exons 12-44, 18-44, 44, 46, 46-47, 46-48, 46-49, 46-51,46-53, 46-55, 46-59, 46-60 of the DMD pre-mRNA of said patient(Aartsma-Rus and van Deutekom, 2007, Antisense Elements (Genetics)Research Focus, 2007 Nova Science Publishers, Inc) as well as from DMDpatients requiring the simultaneous skipping of one of more exons inaddition to exon 45 including but not limited to patients with adeletion of exons 46-50 requiring the co-skipping of exons 45 and 51, orwith a deletion of exons 46-52 requiring the co-skipping of exons 45 and53.

In a preferred method, one or more symptom(s) or characteristic(s) of amyogenic cell or muscle cell from a DMD patient is/are alleviated. Suchsymptoms or characteristics may be assessed at the cellular, tissuelevel or on the patient self.

An alleviation of one or more symptoms or characteristics may beassessed by any of the following assays on a myogenic cell or musclecell from a patient: reduced calcium uptake by muscle cells, decreasedcollagen synthesis, altered morphology, altered lipid biosynthesis,decreased oxidative stress, and/or improved muscle fiber function,integrity, and/or survival. These parameters are usually assessed usingimmunofluorescence and/or histochemical analyses of cross sections ofmuscle biopsies.

The improvement of muscle fiber function, integrity and/or survival mayalso be assessed using at least one of the following assays: adetectable decrease of creatine kinase in blood, a detectable decreaseof necrosis of muscle fibers in a biopsy cross-section of a musclesuspected to be dystrophic, and/or a detectable increase of thehomogeneity of the diameter of muscle fibers in a biopsy cross-sectionof a muscle suspected to be dystrophic. Each of these assays is known tothe skilled person.

Creatine kinase may be detected in blood as described in Hodgetts et al(Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006).A detectable decrease in creatine kinase may mean a decrease of 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%/a, 80%, 90% or more compared to theconcentration of creatine kinase in a same DMD patient before treatment.

A detectable decrease of necrosis of muscle fibers is preferablyassessed in a muscle biopsy, more preferably as described in Hodgetts etal (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16:591-602.2006) using biopsy cross-sections. A detectable decrease ofnecrosis may be a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%/a,80%, 90% or more of the area wherein necrosis has been identified usingbiopsy cross-sections. The decrease is measured by comparison to thenecrosis as assessed in a same DMD patient before treatment.

A detectable increase of the homogeneity of the diameter of musclefibers is preferably assessed in a muscle biopsy cross-section, morepreferably as described in Hodgetts et al (Hodgetts S., et al, (2006),Neuromuscular Disorders, 16: 591-602.2006). The increase is measured bycomparison to the homogeneity of the diameter of muscle fibers in amuscle biopsy cross-section of a same DMD patient before treatment.

An alleviation of one or more symptoms or characteristics may beassessed by any of the following assays on the patient self:prolongation of time to loss of walking, improvement of muscle strength,improvement of the ability to lift weight, improvement of the time takento rise from the floor, improvement in the nine-meter walking time,improvement in the time taken for four-stairs climbing, improvement ofthe leg function grade, improvement of the pulmonary function,improvement of cardiac function, improvement of the quality of life.Each of these assays is known to the skilled person. As an example, thepublication of Manzur at al (Manzur A Y et al, (2008), Glucocorticoidcorticosteroids for Duchenne muscular dystrophy (review), Wileypublishers, The Cochrane collaboration) gives an extensive explanationof each of these assays. For each of these assays, as soon as adetectable improvement or prolongation of a parameter measured in anassay has been found, it will preferably mean that one or more symptomsof Duchenne Muscular Dystrophy has been alleviated in an individualusing a method of the invention. Detectable improvement or prolongationis preferably a statistically significant improvement or prolongation asdescribed in Hodgetts et al (Hodgetts S., et al, (2006), NeuromuscularDisorders, 16: 591-602.2006). Alternatively, the alleviation of one ormore symptom(s) of Duchenne Muscular Dystrophy may be assessed bymeasuring an improvement of a muscle fiber function, integrity and/orsurvival as later defined herein.

A treatment in a method according to the invention may have a durationof at least one week, at least one month, at least several months, atleast one year, at least 2, 3, 4, 5, 6 years or more. The frequency ofadministration of an oligonucleotide, composition, compound of theinvention may depend on several parameters such as the age of thepatient, the type of mutation, the number of molecules (dose), theformulation of said molecule. The frequency may be ranged between atleast once in a two weeks, or three weeks or four weeks or five weeks ora longer time period.

Each molecule or oligonucleotide or equivalent thereof as defined hereinfor use according to the invention may be suitable for directadministration to a cell, tissue and/or an organ in vivo of individualsaffected by or at risk of developing DMD and may be administereddirectly in vivo, ex vivo or in vitro. An oligonucleotide as used hereinmay be suitable for administration to a cell, tissue and/or an organ invivo of individuals affected by or at risk of developing DMD, and may beadministered in vivo, ex vivo or in vitro. Said oligonucleotide may bedirectly or indirectly administrated to a cell, tissue and/or an organin vivo of an individual affected by or at risk of developing DMD, andmay be administered directly or indirectly in vivo, ex vivo or in vitro.As Duchenne muscular dystrophy has a pronounced phenotype in musclecells, it is preferred that said cells are muscle cells, it is furtherpreferred that said tissue is a muscular tissue and/or it is furtherpreferred that said organ comprises or consists of a muscular tissue. Apreferred organ is the heart. Preferably said cells comprise a geneencoding a mutant dystrophin protein. Preferably said cells are cells ofan individual suffering from DMD.

A molecule or oligonucleotide or equivalent thereof can be delivered asis to a cell. When administering said molecule, oligonucleotide orequivalent thereof to an individual, it is preferred that it isdissolved in a solution that is compatible with the delivery method. Forintravenous, subcutaneous, intramuscular, intrathecal and/orintraventricular administration it is preferred that the solution is aphysiological salt solution. Particularly preferred for a method of theinvention is the use of an excipient that will further enhance deliveryof said molecule, oligonucleotide or functional equivalent thereof asdefined herein, to a cell and into a cell, preferably a muscle cell.Preferred excipient are defined in the section entitled “pharmaceuticalcomposition”. In vitro, we obtained very good results usingpolyethylenimine (PEI, ExGen500, MBI Fermentas) as shown in the example.

In a preferred method of the invention, an additional molecule is usedwhich is able to induce and/or promote skipping of a distinct exon ofthe DMD pre-mRNA of a patient. Preferably, the second exon is selectedfrom: exon 7, 44, 46, 51, 53, 59, 67 of the dystrophin pre-mRNA of apatient. Molecules which can be used are depicted in table 2. Preferredmolecules comprise or consist of any of the oligonucleotides asdisclosed in table 2. Several oligonucleotides may also be used incombination. This way, inclusion of two or more exons of a DMD pre-mRNAin mRNA produced from this pre-mRNA is prevented. This embodiment isfurther referred to as double- or multi-exon skipping (Aartsma-Rus A,Janson A A, Kaman W E, et al. Antisense-induced multiexon skipping forDuchenne muscular dystrophy makes more sense. Am J Hum Genet 2004;74(1):83-92, Aartsma-Rus A, Kaman W E, Weij R, den Dunnen J T, van OmmenG J, van Deutekom J C. Exploring the frontiers of therapeutic exonskipping for Duchenne muscular dystrophy by double targeting within oneor multiple exons. Mol Ther 2006; 14(3):401-7). In most casesdouble-exon skipping results in the exclusion of only the two targetedexons from the dystrophin pre-mRNA. However, in other cases it was foundthat the targeted exons and the entire region in between said exons insaid pre-mRNA were not present in the produced mRNA even when otherexons (intervening exons) were present in such region. Thismulti-skipping was notably so for the combination of oligonucleotidesderived from the DMD gene, wherein one oligonucleotide for exon 45 andone oligonucleotide for exon 51 was added to a cell transcribing the DMDgene. Such a set-up resulted in mRNA being produced that did not containexons 45 to 51. Apparently, the structure of the pre-mRNA in thepresence of the mentioned oligonucleotides was such that the splicingmachinery was stimulated to connect exons 44 and 52 to each other.

It is possible to specifically promote the skipping of also theintervening exons by providing a linkage between the two complementaryoligonucleotides. Hence, in one embodiment stretches of nucleotidescomplementary to at least two dystrophin exons are separated by alinking moiety. The at least two stretches of nucleotides are thuslinked in this embodiment so as to form a single molecule.

In case, more than one compounds are used in a method of the invention,said compounds can be administered to an individual in any order. In oneembodiment, said compounds are administered simultaneously (meaning thatsaid compounds are administered within 10 hours, preferably within onehour). This is however not necessary. In another embodiment, saidcompounds are administered sequentially.

Molecule

In a second aspect, there is provided a molecule for use in a method asdescribed in the previous section entitled “Method”. This moleculepreferably comprises or consists of an oligonucleotide, Saidoligonucleotide is preferably an antisense oligonucleotide (AON) orantisense oligoribonucleotide.

It was found by the present investigators that especially exon 45 isspecifically skipped at a high frequency using a molecule that binds toa continuous stretch of at least 21 nucleotides within said exon.Although this effect can be associated with a higher binding affinity ofsaid molecule, compared to a molecule that binds to a continuous stretchof less than 21 nucleotides, there could be other intracellularparameters involved that favor thermodynamic, kinetic, or structuralcharacteristics of the hybrid duplex. In a preferred embodiment, amolecule that binds to a continuous stretch of at least 21, 25, 30, 35,40, 45, 50 nucleotides within said exon is used.

In a preferred embodiment, a molecule or an oligonucleotide of theinvention which comprises a sequence that is complementary to a part ofexon 45 of DMD pre-mRNA is such that the complementary part is at least50% of the length of the oligonucleotide of the invention, morepreferably at least 60%, even more preferably at least 70%, even morepreferably at least 80%, even more preferably at least 90% or even morepreferably at least 95%, or even more preferably 98% and most preferablyup to 100%. “A part of exon 45” preferably means a stretch of at least21 nucleotides. In a most preferred embodiment, an oligonucleotide ofthe invention consists of a sequence that is complementary to part ofexon 45 dystrophin pre-mRNA as defined herein. Alternatively, anoligonucleotide may comprise a sequence that is complementary to part ofexon 45 dystrophin pre-mRNA as defined herein and additional flankingsequences. In a more preferred embodiment, the length of saidcomplementary part of said oligonucleotide is of at least 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides. Several types offlanking sequences may be used. Preferably, additional flankingsequences are used to modify the binding of a protein to said moleculeor oligonucleotide, or to modify a thermodynamic property of theoligonucleotide, more preferably to modify target RNA binding affinity.In another preferred embodiment, additional flanking sequences arecomplementary to sequences of the DMD pre-mRNA which are not present inexon 45. Such flanking sequences are preferably complementary tosequences comprising or consisting of the splice site acceptor or donorconsensus sequences of exon 45. In a preferred embodiment, such flankingsequences are complementary to sequences comprising or consisting ofsequences of an intron of the DMD pre-mRNA which is adjacent to exon 45;i.e. intron 44 or 45. A continuous stretch of at least 21, 25, 30, 35,40, 45, 50 nucleotides within exon 45 is preferably selected from thesequence:

(SEQ ID NO 2) 5′- CCAGGAUGGCAUUGGGCAGCGGCAAACUGUUGUCAGAACAUUGAAUGCAACUGGGGAAGAAAUAAUUCAGCAAUC -3′.

It was found that a molecule that binds to a nucleotide sequencecomprising or consisting of a continuous stretch of at least 21, 25, 30,35, 40, 45, 50 nucleotides of SEQ ID NO. 2 results in highly efficientskipping of exon 45 in a cell provided with this molecule. Moleculesthat bind to a nucleotide sequence comprising a continuous stretch ofless than 21 nucleotides of SEQ ID NO:2 were found to induce exonskipping in a less efficient way than the molecules of the invention.Therefore, in a preferred embodiment, a method is provided wherein amolecule binds to a continuous stretch of at least 21, 25, 30, 35nucleotides within SEQ ID NO:2. Contrary to what was generally thought,the inventors surprisingly found that a higher specificity andefficiency of exon skipping may be reached using an oligonucleotideshaving a length of at least 21 nucleotides. None of the indicatedsequences is derived from conserved parts of splice-junction sites.Therefore, said molecule is not likely to mediate differential splicingof other exons from the DMD pre-mRNA or exons from other genes.

In one embodiment, a molecule of the invention capable of interferingwith the inclusion of exon 45 of the DMD pre-mRNA is a compound moleculethat binds to the specified sequence, or a protein such as anRNA-binding protein or a non-natural zinc-finger protein that has beenmodified to be able to bind to the indicated nucleotide sequence on aRNA molecule. Methods for screening compound molecules that bindspecific nucleotide sequences are for example disclosed inPCT/NL01/00697 and U.S. Pat. No. 6,875,736, which are herein enclosed byreference. Methods for designing RNA-binding Zinc-finger proteins thatbind specific nucleotide sequences are disclosed by Friesen and Darby,Nature Structural Biology 5: 543-546 (1998) which is herein enclosed byreference.

In a further embodiment, a molecule of the invention capable ofinterfering with the inclusion of exon 45 of the DMD pre-mRNA comprisesan antisense oligonucleotide that is complementary to and can base-pairwith the coding strand of the pre-mRNA of the DMD gene. Said antisenseoligonucleotide preferably contains a RNA residue, a DNA residue, and/ora nucleotide analogue or equivalent, as will be further detailed hereinbelow.

A preferred molecule of the invention comprises a nucleotide-based ornucleotide or an antisense oligonucleotide sequence of between 21 and 50nucleotides or bases, more preferred between 21 and 40 nucleotides, morepreferred between 21 and 30 nucleotides, such as 21 nucleotides, 22nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides or 50nucleotides.

A most preferred molecule of the invention comprises a nucleotide-basedsequence of 25 nucleotides.

In a preferred embodiment, a molecule of the invention binds to acontinuous stretch of or is complementary to or is antisense to at leasta continuous stretch of at least 21 nucleotides within the nucleotidesequence SEQ ID NO:2.

In a certain embodiment, the invention provides a molecule comprising orconsisting of an antisense nucleotide sequence selected from theantisense nucleotide sequences as depicted in Table 1, except SEQ IDNO:68. A molecule of the invention that is antisense to the sequence ofSEQ ID NO 2, which is present in exon 45 of the DMD gene preferablycomprises or consists of the antisense nucleotide sequence of SEQ ID NO3; SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ IDNO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO64, SEQ ID NO 65, SEQ ID NO 66 and/or SEQ ID NO:67.

In a more preferred embodiment, the invention provides a moleculecomprising or consisting of the antisense nucleotide sequence of SEQ IDNO 3; SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and/or SEQ IDNO 8.

In a most preferred embodiment, the invention provides a moleculecomprising or consisting of the antisense nucleotide sequence of SEQ IDNO 3. It was found that this molecule is very efficient in modulatingsplicing of exon 45 of the DMD pre-mRNA in a muscle cell.

A nucleotide sequence of a molecule of the invention may contain a RNAresidue, a DNA residue, a nucleotide analogue or equivalent as will befurther detailed herein below. In addition, a molecule of the inventionmay encompass a functional equivalent of a molecule of the invention asdefined herein.

It is preferred that a molecule of the invention comprises a or at leastone residue that is modified to increase nuclease resistance, and/or toincrease the affinity of the antisense nucleotide for the targetsequence. Therefore, in a preferred embodiment, an antisense nucleotidesequence comprises a or at least one nucleotide analogue or equivalent,wherein a nucleotide analogue or equivalent is defined as a residuehaving a modified base, and/or a modified backbone, and/or a non-naturalinternucleoside linkage, or a combination of these modifications.

In a preferred embodiment, a nucleotide analogue or equivalent comprisesa modified backbone. Examples of such backbones are provided bymorpholino backbones, carbamate backbones, siloxane backbones, sulfide,sulfoxide and sulfone backbones, formacetyl and thioformacetylbackbones, methyleneformacetyl backbones, riboacetyl backbones, alkenecontaining backbones, sulfamate, sulfonate and sulfonamide backbones,methyleneimino and methylenehydrazino backbones, and amide backbones.Phosphorodiamidate morpholino oligomers are modified backboneoligonucleotides that have previously been investigated as antisenseagents. Morpholino oligonucleotides have an uncharged backbone in whichthe deoxyribose sugar of DNA is replaced by a six membered ring and thephosphodiester linkage is replaced by a phosphorodiamidate linkage.Morpholino oligonucleotides are resistant to enzymatic degradation andappear to function as antisense agents by arresting translation orinterfering with pre-mRNA splicing rather than by activating RNase H.Morpholino oligonucleotides have been successfully delivered to tissueculture cells by methods that physically disrupt the cell membrane, andone study comparing several of these methods found that scrape loadingwas the most efficient method of delivery; however, because themorpholino backbone is uncharged, cationic lipids are not effectivemediators of morpholino oligonucleotide uptake in cells. A recent reportdemonstrated triplex formation by a morpholino oligonucleotide and,because of the non-ionic backbone, these studies showed that themorpholino oligonucleotide was capable of triplex formation in theabsence of magnesium.

It is further preferred that the linkage between a residue in a backbonedoes not include a phosphorus atom, such as a linkage that is formed byshort chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.

A preferred nucleotide analogue or equivalent comprises a PeptideNucleic Acid (PNA), having a modified polyamide backbone (Nielsen, etal. (1991) Science 254, 1497-1500). PNA-based molecules are true mimicsof DNA molecules in terms of base-pair recognition. The backbone of thePNA is composed of N-(2-aminoethyl)-glycine units linked by peptidebonds, wherein the nucleobases are linked to the backbone by methylenecarbonyl bonds. An alternative backbone comprises a one-carbon extendedpyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem. Commun,495-497). Since the backbone of a PNA molecule contains no chargedphosphate groups, PNA-RNA hybrids are usually more stable than RNA-RNAor RNA-DNA hybrids, respectively (Egholm et al (1993) Nature 365,566-568).

A further preferred backbone comprises a morpholino nucleotide analog orequivalent, in which the ribose or deoxyribose sugar is replaced by a6-membered morpholino ring. A most preferred nucleotide analog orequivalent comprises a phosphorodiamidate morpholino oligomer (PMO), inwhich the ribose or deoxyribose sugar is replaced by a 6-memberedmorpholino ring, and the anionic phosphodiester linkage between adjacentmorpholino rings is replaced by a non-ionic phosphorodiamidate linkage.

In yet a further embodiment, a nucleotide analogue or equivalent of theinvention comprises a substitution of at least one of the non-bridgingoxygens in the phosphodiester linkage. This modification slightlydestabilizes base-pairing but adds significant resistance to nucleasedegradation. A preferred nucleotide analogue or equivalent comprisesphosphorothioate, chiral phosphorothioate, phosphorodithioate,phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl andother alkyl phosphonate including 3′-alkylene phosphonate, 5′-alkylenephosphonate and chiral phosphonate, phosphinate, phosphoramidateincluding 3′-amino phosphoramidate and aminoalkylphosphoramidate,thionophosphoramidate, thionoalkylphosphonate,thionoalkylphosphotriester, selenophosphate or boranophosphate.

A further preferred nucleotide analogue or equivalent of the inventioncomprises one or more sugar moieties that are mono- or disubstituted atthe 2′, 3′ and/or 5′ position such as a —OH; —F; substituted orunsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl,alkynyl, alkaryl, allyl, aryl, or aralkyl, that may be interrupted byone or more heteroatoms; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-,S- or N-alkynyl; O-, S-, or N-allyl; O-alkyl-O-alkyl, -methoxy,-aminopropoxy; aminoxy, methoxyethoxy; -dimethylaminooxyethoxy; and-dimethylaminoethoxyethoxy. The sugar moiety can be a pyranose orderivative thereof, or a deoxypyranose or derivative thereof, preferablya ribose or a derivative thereof, or deoxyribose or derivative thereof.Such preferred derivatized sugar moieties comprise Locked Nucleic Acid(LNA), in which the 2′-carbon atom is linked to the 3′ or 4′ carbon atomof the sugar ring thereby forming a bicyclic sugar moiety. A preferredLNA comprises 2′-O,4′-C-ethylene-bridged nucleic acid (Morita et al.2001. Nucleic Acid Res Supplement No. 1: 241-242). These substitutionsrender the nucleotide analogue or equivalent RNase H and nucleaseresistant and increase the affinity for the target RNA.

It is understood by a skilled person that it is not necessary for allpositions in an antisense oligonucleotide to be modified uniformly. Inaddition, more than one of the aforementioned analogues or equivalentsmay be incorporated in a single antisense oligonucleotide or even at asingle position within an antisense oligonucleotide. In certainembodiments, an antisense oligonucleotide of the invention has at leasttwo different types of analogues or equivalents.

A preferred antisense oligonucleotide according to the inventioncomprises a 2′-O-alkyl phosphorothioate antisense oligonucleotide, suchas 2′-O-methyl modified ribose (RNA), 2′-O-ethyl modified ribose,2′-O-propyl modified ribose, and/or substituted derivatives of thesemodifications such as halogenated derivatives.

A most preferred antisense oligonucleotide according to the inventioncomprises a 2′-O-methyl phosphorothioate ribose.

A functional equivalent of a molecule of the invention may be defined asan oligonucleotide as defined herein wherein an activity of saidfunctional equivalent is retained to at least some extent. Preferably,an activity of said functional equivalent is inducing exon 45 skippingand providing a functional dystrophin protein. Said activity of saidfunctional equivalent is therefore preferably assessed by detection ofexon 45 skipping and quantifying the amount of a functional dystrophinprotein. A functional dystrophin is herein preferably defined as being adystrophin able to bind actin and members of the DGC protein complex.The assessment of said activity of an oligonucleotide is preferably doneby RT-PCR or by immunofluorescence or Western blot analysis. Saidactivity is preferably retained to at least some extent when itrepresents at least 50%, or at least 60%, or at least 70% or at least80% or at least 90% or at least 95% or more of corresponding activity ofsaid oligonucleotide the functional equivalent derives from. Throughoutthis application, when the word oligonucleotide is used it may bereplaced by a functional equivalent thereof as defined herein.

It will also be understood by a skilled person that distinct antisenseoligonucleotides can be combined for efficiently skipping of exon 45 ofthe human DMD pre-mRNA. In a preferred embodiment, a combination of atleast two antisense oligonucleotides are used in a method of theinvention, such as two distinct antisense oligonucleotides, threedistinct antisense oligonucleotides, four distinct antisenseoligonucleotides, or five distinct antisense oligonucleotides or evenmore. It is also encompassed by the present invention to combine severaloligonucleotides or molecules as depicted in table 1 except SEQ IDNO:68.

An antisense oligonucleotide can be linked to a moiety that enhancesuptake of the antisense oligonucleotide in cells, preferably myogeniccells or muscle cells. Examples of such moieties are cholesterols,carbohydrates, vitamins, biotin, lipids, phospholipids, cell-penetratingpeptides including but not limited to antennapedia, TAT, transportan andpositively charged amino acids such as oligoarginine, poly-arginine,oligolysine or polylysine, antigen-binding domains such as provided byan antibody, a Fab fragment of an antibody, or a single chain antigenbinding domain such as a cameloid single domain antigen-binding domain.

A preferred antisense oligonucleotide comprises a peptide-linked PMO.

A preferred antisense oligonucleotide comprising one or more nucleotideanalogs or equivalents of the invention modulates splicing in one ormore muscle cells, including heart muscle cells, upon systemic delivery.In this respect, systemic delivery of an antisense oligonucleotidecomprising a specific nucleotide analog or equivalent might result intargeting a subset of muscle cells, while an antisense oligonucleotidecomprising a distinct nucleotide analog or equivalent might result intargeting of a different subset of muscle cells. Therefore, in oneembodiment it is preferred to use a combination of antisenseoligonucleotides comprising different nucleotide analogs or equivalentsfor modulating skipping of exon 45 of the human DMD pre-mRNA.

A cell can be provided with a molecule capable of interfering withessential sequences that result in highly efficient skipping of exon 45of the human DMD pre-mRNA by plasmid-derived antisense oligonucleotideexpression or viral expression provided by viral-based vector. Such aviral-based vector comprises an expression cassette that drivesexpression of an antisense molecule as defined herein. Preferredvirus-based vectors include adenovirus- or adeno-associated virus-basedvectors. Expression is preferably driven by a polymerase III promoter,such as a U1, a U6, or a U7 RNA promoter. A muscle or myogenic cell canbe provided with a plasmid for antisense oligonucleotide expression byproviding the plasmid in an aqueous solution. Alternatively, a plasmidcan be provided by transfection using known transfection agentia suchas, for example, LipofectAMINE™ 2000 (Invitrogen) or polyethyleneimine(PEI; ExGen500 (MBI Fermentas)), or derivatives thereof.

One preferred antisense oligonucleotide expression system is anadenovirus associated virus (AAV)-based vector. Single chain and doublechain AAV-based vectors have been developed that can be used forprolonged expression of small antisense nucleotide sequences for highlyefficient skipping of exon 45 of the DMD pre-mRNA.

A preferred AAV-based vector comprises an expression cassette that isdriven by a polymerase III-promoter (Pol III). A preferred Pol IIIpromoter is, for example, a U1, a U6, or a U7 RNA promoter.

The invention therefore also provides a viral-based vector, comprising aPol III-promoter driven expression cassette for expression of one ormore antisense sequences of the invention for inducing skipping of exon45 of the human DMD pre-mRNA.

Pharmaceutical Composition

If required, a molecule or a vector expressing an antisenseoligonucleotide of the invention can be incorporated into apharmaceutically active mixture or composition by adding apharmaceutically acceptable carrier.

Therefore, in a further aspect, the invention provides a composition,preferably a pharmaceutical composition comprising a molecule comprisingan antisense oligonucleotide according to the invention, and/or aviral-based vector expressing the antisense sequence(s) according to theinvention and a pharmaceutically acceptable carrier.

A preferred pharmaceutical composition comprises a molecule as definedherein and/or a vector as defined herein, and a pharmaceuticalacceptable carrier or excipient, optionally combined with a moleculeand/or a vector which is able to modulate skipping of exon 7, 44, 46,51, 53, 59, 67 of the DMD pre-mRNA.

Preferred excipients include excipients capable of forming complexes,vesicles and/or liposomes that deliver such a molecule as definedherein, preferably an oligonucleotide complexed or trapped in a vesicleor liposome through a cell membrane. Many of these excipients are knownin the art. Suitable excipients comprise polyethylenimine andderivatives, or similar cationic polymers, including polypropyleneimineor polyethylenimine copolymers (PECs) and derivatives, syntheticamphiphils, Lipofectin™, DOTAP and/or viral capsid proteins that arecapable of self assembly into particles that can deliver such molecule,preferably an oligonucleotide as defined herein to a cell, preferably amuscle cell. Such excipients have been shown to efficiently deliver(oligonucleotide such as antisense) nucleic acids to a wide variety ofcultured cells, including muscle cells. We obtained very good resultsusing polyethylenimine (PEI, ExGen500, MBI Fermentas) as shown in theexample. Their high transfection potential is combined with an exceptedlow to moderate toxicity in terms of overall cell survival. The ease ofstructural modification can be used to allow further modifications andthe analysis of their further (in vivo) nucleic acid transfercharacteristics and toxicity.

Lipofectin represents an example of a liposomal transfection agent. Itconsists of two lipid components, a cationic lipid N-[1-(2,3dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAPwhich is the methylsulfate salt) and a neutral lipiddioleoylphosphatidylethanolamine (DOPE). The neutral component mediatesthe intracellular release. Another group of delivery systems arepolymeric nanoparticles.

Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, whichare well known as DNA transfection reagent can be combined withbutylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulatecationic nanoparticles that can deliver a molecule or a compound asdefined herein, preferably an oligonucleotide across cell membranes intocells.

In addition to these common nanoparticle materials, the cationic peptideprotamine offers an alternative approach to formulate a compound asdefined herein, preferably an oligonucleotide as colloids. Thiscolloidal nanoparticle system can form so called proticles, which can beprepared by a simple self-assembly process to package and mediateintracellular release of a compound as defined herein, preferably anoligonucleotide. The skilled person may select and adapt any of theabove or other commercially available alternative excipients anddelivery systems to package and deliver a compound as defined herein,preferably an oligonucleotide for use in the current invention todeliver said compound for the treatment of Duchenne Muscular Dystrophyin humans.

In addition, a compound as defined herein, preferably an oligonucleotidecould be covalently or non-covalently linked to a targeting ligandspecifically designed to facilitate the uptake in to the cell, cytoplasmand/or its nucleus. Such ligand could comprise (i) a compound (includingbut not limited to peptide(-like) structures) recognising cell, tissueor organ specific elements facilitating cellular uptake and/or (ii) achemical compound able to facilitate the uptake in to cells and/or theintracellular release of an a compound as defined herein, preferably anoligonucleotide from vesicles, e.g. endosomes or lysosomes.

Therefore, in a preferred embodiment, a compound as defined herein,preferably an oligonucleotide are formulated in a medicament which isprovided with at least an excipient and/or a targeting ligand fordelivery and/or a delivery device of said compound to a cell and/orenhancing its intracellular delivery. Accordingly, the invention alsoencompasses a pharmaceutically acceptable composition comprising acompound as defined herein, preferably an oligonucleotide and furthercomprising at least one excipient and/or a targeting ligand for deliveryand/or a delivery device of said compound to a cell and/or enhancing itsintracellular delivery.

It is to be understood that a molecule or compound or oligonucleotidemay not be formulated in one single composition or preparation.Depending on their identity, the skilled person will know which type offormulation is the most appropriate for each compound.

In a preferred embodiment, an in vitro concentration of a molecule or anoligonucleotide as defined herein, which is ranged between 0.1 nM and 1μM is used. More preferably, the concentration used is ranged between0.3 to 400 nM, even more preferably between 1 to 200 nM. molecule or anoligonucleotide as defined herein may be used at a dose which is rangedbetween 0.1 and 20 mg/kg, preferably 0.5 and 10 mg/kg. If severalmolecules or oligonucleotides are used, these concentrations may referto the total concentration of oligonucleotides or the concentration ofeach oligonucleotide added. The ranges of concentration ofoligonucleotide(s) as given above are preferred concentrations for invitro or ex vivo uses. The skilled person will understand that dependingon the oligonucleotide(s) used, the target cell to be treated, the genetarget and its expression levels, the medium used and the transfectionand incubation conditions, the concentration of oligonucleotide(s) usedmay further vary and may need to be optimised any further.

More preferably, a compound preferably an oligonucleotide and an adjunctcompound to be used in the invention to prevent, treat DMD aresynthetically produced and administered directly to a cell, a tissue, anorgan and/or patients in formulated form in a pharmaceuticallyacceptable composition or preparation. The delivery of a pharmaceuticalcomposition to the subject is preferably carried out by one or moreparenteral injections, e.g. intravenous and/or subcutaneous and/orintramuscular and/or intrathecal and/or intraventricularadministrations, preferably injections, at one or at multiple sites inthe human body.

Use

In yet a further aspect, the invention provides the use of an antisenseoligonucleotide or molecule according to the invention, and/or aviral-based vector that expresses one or more antisense sequencesaccording to the invention and/or a pharmaceutical composition, forinducing and/or promoting splicing of the DMD pre-mRNA. The splicing ispreferably modulated in a human myogenic cell or a muscle cell in vitro.More preferred is that splicing is modulated in human a myogenic cell ormuscle cell in vivo.

Accordingly, the invention further relates to the use of the molecule asdefined herein and/or the vector as defined herein and/or or thepharmaceutical composition as defined herein for inducing and/orpromoting splicing of the DMD pre-mRNA or for the preparation of amedicament for the treatment of a DMD patient.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition the verb “to consist” may be replaced by“to consist essentially of” meaning that a molecule or a viral-basedvector or a composition as defined herein may comprise additionalcomponent(s) than the ones specifically identified, said additionalcomponent(s) not altering the unique characteristic of the invention. Inaddition, reference to an element by the indefinite article “a” or “an”does not exclude the possibility that more than one of the element ispresent, unless the context clearly requires that there be one and onlyone of the elements. The indefinite article “a” or “an” thus usuallymeans “at least one”.

Each embodiment as identified herein may be combined together unlessotherwise indicated. All patent and literature references cited in thepresent specification are hereby incorporated by reference in theirentirety.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

LEGENDS TO THE FIGURE

FIG. 1. In human control myotubes, a series of AONs (PS220 to PS225; SEQID NO: 3 to 8), all binding to a continuous stretch of at least 21nucleotides within a specific sequence of exon 45 (i.e. SEQ ID NO:2),were tested at two different concentrations (200 and 500 nM). All sixAONs were effective in inducing specific exon 45 skipping, as confirmedby sequence analysis (not shown). PS220 (SEQ ID NO:3) however,reproducibly induced highest levels of exon 45 skipping (see FIG. 2).(NT: non-treated cells, M: size marker).

FIG. 2. In human control myotubes, 25-mer PS220 (SEQ ID NO: 3) wastested at increasing concentration. Levels of exon 45 skipping of up to75% (at 400 nM) were observed reproducibly, as assessed by AgilentLabChip Analysis.

FIG. 3. In human control myotubes, the efficiencies of a “short” 17-merAON45-5 (SEQ ID NO:68) and its overlapping “long” 25-mer counterpartPS220 were directly compared at 200 nM and 500 nM. PS220 was markedlymore efficient at both concentrations: 63% when compared to 3% obtainedwith 45-5. (NT: non-treated cells, M: size marker).

EXAMPLES Examples 1 and 2

Materials and Methods

AON design was based on (partly) overlapping open secondary structuresof the target exon RNA as predicted by the m-fold program (Zuker, M.(2003) Mfold web server for nucleic acid folding and hybridizationprediction. Nucleic Acids Res., 31, 3406-3415), and on (partly)overlapping putative SR-protein binding sites as predicted by numeroussoftware programs such as ESEfinder (Cartegni, L. et al. (2003)ESEfinder: A web resource to identify exonic splicing enhancers. NucleicAcids Res, 31, 3568-71; Smith, P. J. et al. (2006) An increasedspecificity score matrix for the prediction of SF2/ASF-specific exonicsplicing enhancers. Hum. Mol. Genet., 15, 2490-2508) that predictsbinding sites for the four most abundant SR proteins (SF2/ASF, SC35,SRp40 and SRp55). AONs were synthesized by Prosensa Therapeutics B.V.(Leiden, Netherlands), and contain 2′-O-methyl RNA and full-lengthphosphorothioate (PS) backbones.

Tissue Culturing, Transfection and RT-PCR Analysis

Myotube cultures derived from a healthy individual (“human control”)were obtained as described previously (Aartsma-Rus et al. Hum Mol Genet2003; 12(8): 907-14). For the screening of AONs, myotube cultures weretransfected with 0 to 500 nM of each AON. The transfection reagentpolyethylenimine (PEI, ExGen500 MBI Fermentas) was used according tomanufacturer's instructions, with 2 μl PEI per μg AON. Exon skippingefficiencies were determined by nested RT-PCR analysis using primers inthe exons flanking exon 45. PCR fragments were isolated from agarosegels for sequence verification. For quantification, the PCR productswere analyzed using the Agilent DNA 1000 LabChip Kit and the Agilent2100 bioanalyzer (Agilent Technologies, USA).

Results

A series of AONs targeting sequences within SEQ ID NO:2 within exon 45were designed and tested in normal myotube cultures, by transfection andsubsequent RT-PCR and sequence analysis of isolated RNA. PS220 (SEQ IDNO: 3) reproducibly induced highest levels of exon 45 skipping, whencompared to PS221-PS225 (FIG. 1). High levels of exon 45 skipping of upto 75% were already obtained at 400 nM PS220 (FIG. 2). In a directcomparison, PS220 (a 25-mer) was reproducibly more efficient in inducingexon 45 skipping than its shorter 17-mer counterpart AON 45-5 (SEQ IDNO: 68; previously published as h45AON5 (Aartsma-Rus et al. Am J HumGenet 2004; 74: 83-92)), at both AON concentrations of 200 nM and 500 nMand with 63% versus 3% respectively at 500 nM (FIG. 3). This result isprobably due to the fact that the extended length of PS220, in factcompletely overlapping AON 45-5, increases the free energy of theAON-target complex such that the efficiency of inducing exon 45 skippingis also increased.

TABLE 1 AONs in exon 45 SEQ ID NO 3 UUUGCCGCUGCCCAAUGCCAUCCUG (PS220)SEQ ID NO 4 AUUCAAUGUUCUGACAACAGUUUGC (PS221) SEQ ID NO 5CCAGUUGCAUUCAAUGUUCUGACAA (PS222) SEQ ID NO 6 CAGUUGCAUUCAAUGUUCUGAC(PS223) SEQ ID NO 7 AGUUGCAUUCAAUGUUCUGA (PS224) SEQ ID NO 8GAUUGCUGAAUUAUUUCUUCC (PS225) SEQ ID NO 9 GAUUGCUGAAUUAUUUCUUCCCCAGSEQ ID NO 10 AUUGCUGAAUUAUUUCUUCCCCAGU SEQ ID NO 11UUGCUGAAUUAUUUCUUCCCCAGUU SEQ ID NO 12 UGCUGAAUUAUUUCUUCCCCAGUUGSEQ ID NO 13 GCUGAAUUAUUUCUUCCCCAGUUGC SEQ ID NO 14CUGAAUUAUUUCUUCCCCAGUUGCA SEQ ID NO 15 UGAAUUAUUUCUUCCCCAGUUGCAUSEQ ID NO 16 GAAUUAUUUCUUCCCCAGUUGCAUU SEQ ID NO 17AAUUAUUUCUUCCCCAGUUGCAUUC SEQ ID NO 18 AUUAUUUCUUCCCCAGUUGCAUUCASEQ ID NO 19 UUAUUUCUUCCCCAGUUGCAUUCAA SEQ ID NO 20UAUUUCUUCCCCAGUUGCAUUCAAU SEQ ID NO 21 AUUUCUUCCCCAGUUGCAUUCAAUGSEQ ID NO 22 UUUCUUCCCCAGUUGCAUUCAAUGU SEQ ID NO 23UUCUUCCCCAGUUGCAUUCAAUGUU SEQ ID NO 24 UCUUCCCCAGUUGCAUUCAAUGUUCSEQ ID NO 25 CUUCCCCAGUUGCAUUCAAUGUUCU SEQ ID NO 26UUCCCCAGUUGCAUUCAAUGUUCUG SEQ ID NO 27 UCCCCAGUUGCAUUCAAUGUUCUGASEQ ID NO 28 CCCCAGUUGCAUUCAAUGUUCUGAC SEQ ID NO 29CCCAGUUGCAUUCAAUGUUCUGACA SEQ ID NO 30 CCAGUUGCAUUCAAUGUUCUGACAASEQ ID NO 31 CAGUUGCAUUCAAUGUUCUGACAAC SEQ ID NO 32AGUUGCAUUCAAUGUUCUGACAACA SEQ ID NO 33 UCC UGU AGA AUA CUG GCA UCSEQ ID NO 34 UGC AGA CCU CCU GCC ACC GCA GAU UCA SEQ ID NO 35UUGCAGACCUCCUGCCACCGCAGAUUC AGGCUUC SEQ ID NO 36GUUGCAUUCAAUGUUCUGACAACAG SEQ ID NO 37 UUGCAUUCAAUGUUCUGACAACAGUSEQ ID NO 38 UGCAUUCAAUGUUCUGACAACAGUU SEQ ID NO 39GCAUUCAAUGUUCUGACAACAGUUU SEQ ID NO 40 CAUUCAAUGUUCUGACAACAGUUUGSEQ ID NO 41 AUUCAAUGUUCUGACAACAGUUUGC SEQ ID NO 42UCAAUGUUCUGACAACAGUUUGCCG SEQ ID NO 43 CAAUGUUCUGACAACAGUUUGCCGCSEQ ID NO 44 AAUGUUCUGACAACAGUUUGCCGCU SEQ ID NO 45AUGUUCUGACAACAGUUUGCCGCUG SEQ ID NO 46 UGUUCUGACAACAGUUUGCCGCUGCSEQ ID NO 47 GUUCUGACAACAGUUUGCCGCUGCC SEQ ID NO 48UUCUGACAACAGUUUGCCGCUGCCC SEQ ID NO 49 UCUGACAACAGUUUGCCGCUGCCCASEQ ID NO 50 CUGACAACAGUUUGCCGCUGCCCAA SEQ ID NO 51UGACAACAGUUUGCCGCUGCCCAAU SEQ ID NO 52 GACAACAGUUUGCCGCUGCCCAAUGSEQ ID NO 53 ACAACAGUUUGCCGCUGCCCAAUGC SEQ ID NO 54CAACAGUUUGCCGCUGCCCAAUGCC SEQ ID NO 55 AACAGUUUGCCGCUGCCCAAUGCCASEQ ID NO 56 ACAGUUUGCCGCUGCCCAAUGCCAU SEQ ID NO 57CAGUUUGCCGCUGCCCAAUGCCAUC SEQ ID NO 58 AGUUUGCCGCUGCCCAAUGCCAUCCSEQ ID NO 59 GUUUGCCGCUGCCCAAUGCCAUCCU SEQ ID NO 60UUUGCCGCUGCCCAAUGCCAUCCUG SEQ ID NO 61 UUGCCGCUGCCCAAUGCCAUCCUGGSEQ ID NO 62 UGCCGCUGCCCAAUGCCAUCCUGGA SEQ ID NO 63GCCGCUGCCCAAUGCCAUCCUGGAG SEQ ID NO 64 CCGCUGCCCAAUGCCAUCCUGGAGUSEQ ID NO 65 CGCUGCCCAAUGCCAUCCUGGAGUU SEQ ID NO 66UGU UUU UGA GGA UUG CUG AA SEQ ID NO 67 UGUUCUGACAACAGUUUGCCGCUGCCCAAUGCCAUCCUGG SEQ ID NO 68 GCCCAAUGCCAUCCUGG (45-5)

TABLE 2 AONs in exons 51, 53, 7, 44, 46, 59, and 67 DMD Gene Exon 51SEQ ID NO 69 AGAGCAGGUACCUCCAACAUCAAGG SEQ ID NO 70GAGCAGGUACCUCCAACAUCAAGGA SEQ ID NO 71 AGCAGGUACCUCCAACAUCAAGGAASEQ ID NO 72 GCAGGUACCUCCAACAUCAAGGAAG SEQ ID NO 73CAGGUACCUCCAACAUCAAGGAAGA SEQ ID NO 74 AGGUACCUCCAACAUCAAGGAAGAUSEQ ID NO 75 GGUACCUCCAACAUCAAGGAAGAUG SEQ ID NO 76GUACCUCCAACAUCAAGGAAGAUGG SEQ ID NO 77 UACCUCCAACAUCAAGGAAGAUGGCSEQ ID NO 78 ACCUCCAACAUCAAGGAAGAUGGCA SEQ ID NO 79CCUCCAACAUCAAGGAAGAUGGCAU SEQ ID NO 80 CUCCAACAUCAAGGAAGAUGGCAUUSEQ ID NO 81 CUCCAACAUCAAGGAAGAUGGCAUUUCUAG SEQ ID NO 82UCCAACAUCAAGGAAGAUGGCAUUU SEQ ID NO 83 CCAACAUCAAGGAAGAUGGCAUUUCSEQ ID NO 84 CAACAUCAAGGAAGAUGGCAUUUCU SEQ ID NO 85AACAUCAAGGAAGAUGGCAUUUCUA SEQ ID NO 86 ACAUCAAGGAAGAUGGCAUUUCUAGSEQ ID NO 87 ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG SEQ ID NO 88ACAUCAAGGAAGAUGGCAUUUCUAG SEQ ID NO 89 CAUCAAGGAAGAUGGCAUUUCUAGUSEQ ID NO 90 AUCAAGGAAGAUGGCAUUUCUAGUU SEQ ID NO 91UCAAGGAAGAUGGCAUUUCUAGUUU SEQ ID NO 92 UCAAGGAAGAUGGCAUUUCU SEQ ID NO 93CAAGGAAGAUGGCAUUUCUAGUUUG SEQ ID NO 94 AAGGAAGAUGGCAUUUCUAGUUUGGSEQ ID NO 95 AGGAAGAUGGCAUUUCUAGUUUGGA SEQ ID NO 96GGAAGAUGGCAUUUCUAGUUUGGAG SEQ ID NO 97 GAAGAUGGCAUUUCUAGUUUGGAGASEQ ID NO 98 AAGAUGGCAUUUCUAGUUUGGAGAU SEQ ID NO 99AGAUGGCAUUUCUAGUUUGGAGAUG SEQ ID NO 100 GAUGGCAUUUCUAGUUUGGAGAUGGSEQ ID NO 101 AUGGCAUUUCUAGUUUGGAGAUGGC SEQ ID NO 102UGGCAUUUCUAGUUUGGAGAUGGCA SEQ ID NO 103 GGCAUUUCUAGUUUGGAGAUGGCAGSEQ ID NO 104 GCAUUUCUAGUUUGGAGAUGGCAGU SEQ ID NO 105CAUUUCUAGUUUGGAGAUGGCAGUU SEQ ID NO 106 AUUUCUAGUUUGGAGAUGGCAGUUUSEQ ID NO 107 UUUCUAGUUUGGAGAUGGCAGUUUC SEQ ID NO 108UUCUAGUUUGGAGAUGGCAGUUUCC DMD Gene Exon 53 SEQ ID NO 109CCAUUGUGUUGAAUCCUUUAACAUU SEQ ID NO 110 CCAUUGUGUUGAAUCCUUUAACSEQ ID NO 111 AUUGUGUUGAAUCCUUUAAC SEQ ID NO 112 CCUGUCCUAAGACCUGCUCASEQ ID NO 113 CUUUUGGAUUGCAUCUACUGUAUAG SEQ ID NO 114CAUUCAACUGUUGCCUCCGGUUCUG SEQ ID NO 115 CUGUUGCCUCCGGUUCUGAAGGUGSEQ ID NO 116 CAUUCAACUGUUGCCUCCGGUUCUGAAGGUG SEQ ID NO 117CUGAAGGUGUUCUUGUACUUCAUCC SEQ ID NO 118 UGUAUAGGGACCCUCCUUCCAUGACUCSEQ ID NO 119 AUCCCACUGAUUCUGAAUUC SEQ ID NO 120 UUGGCUCUGGCCUGUCCUAAGASEQ ID NO 121 AAGACCUGCUCAGCUUCUUCCUUAGCUUCCAGCCA DMD Gene Exon 7SEQ ID NO 122 UGCAUGUUCCAGUCGUUGUGUGG SEQ ID NO 123CACUAUUCCAGUCAAAUAGGUCUGG SEQ ID NO 124 AUUUACCAACCUUCAGGAUCGAGUASEQ ID NO 125 GGCCUAAAACACAUACACAUA DMD Gene Exon 44 SEQ ID NO 126UCAGCUUCUGUUAGCCACUG SEQ ID NO 127 UUCAGCUUCUGUUAGCCACU SEQ ID NO 128UUCAGCUUCUGUUAGCCACUG SEQ ID NO 129 UCAGCUUCUGUUAGCCACUGA SEQ ID NO 130UUCAGCUUCUGUUAGCCACUGA SEQ ID NO 131 UCAGCUUCUGUUAGCCACUGA SEQ ID NO 132UUCAGCUUCUGUUAGCCACUGA SEQ ID NO 133 UCAGCUUCUGUUAGCCACUGAUSEQ ID NO 134 UUCAGCUUCUGUUAGCCACUGAU SEQ ID NO 135UCAGCUUCUGUUAGCCACUGAUU SEQ ID NO 136 UUCAGCUUCUGUUAGCCACUGAUUSEQ ID NO 137 UCAGCUUCUGUUAGCCACUGAUUA SEQ ID NO 138UUCAGCUUCUGUUAGCCACUGAUA SEQ ID NO 139 UCAGCUUCUGUUAGCCACUGAUUAASEQ ID NO 140 UUCAGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 141UCAGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 142 UUCAGCUUCUGUUAGCCACUGAUUAAASEQ ID NO 143 CAGCUUCUGUUAGCCACUG SEQ ID NO 144 CAGCUUCUGUUAGCCACUGAUSEQ ID NO 145 AGCUUCUGUUAGCCACUGAUU SEQ ID NO 146 CAGCUUCUGUUAGCCACUGAUUSEQ ID NO 147 AGCUUCUGUUAGCCACUGAUUA SEQ ID NO 148CAGCUUCUGUUAGCCACUGAUUA SEQ ID NO 149 AGCUUCUGUUAGCCACUGAUUAASEQ ID NO 150 CAGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 151AGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 152 CAGCUUCUGUUAGCCACUGAUUAAASEQ ID NO 153 AGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 154AGCUUCUGUUAGCCACUGAU SEQ ID NO 155 GCUUCUGUUAGCCACUGAUU SEQ ID NO 156AGCUUCUGUUAGCCACUGAUU SEQ ID NO 157 GCUUCUGUUAGCCACUGAUUA SEQ ID NO 158AGCUUCUGUUAGCCACUGAUUA SEQ ID NO 159 GCUUCUGUUAGCCACUGAUUAASEQ ID NO 160 AGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 161GCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 162 AGCUUCUGUUAGCCACUGAUUAAASEQ ID NO 163 GCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 164CCAUUUGUAUUUAGCAUGUUCCC SEQ ID NO 165 AGAUACCAUUUGUAUUUAGC SEQ ID NO 166GCCAUUUCUCAACAGAUCU SEQ ID NO 167 GCCAUUUCUCAACAGAUCUGUCA SEQ ID NO 168AUUCUCAGGAAUUUGUGUCUUUC SEQ ID NO 169 UCUCAGGAAUUUGUGUCUUUCSEQ ID NO 170 GUUCAGCUUCUGUUAGCC SEQ ID NO 171 CUGAUUAAAUAUCUUUAUAU CSEQ ID NO 172 GCCGCCAUUUCUCAACAG SEQ ID NO 173 GUAUUUAGCAUGUUCCCASEQ ID NO 174 CAGGAAUUUGUGUCUUUC DMD Gene Exon 46 SEQ ID NO 175GCUUUUCUUUUAGUUGCUGCUCUUU SEQ ID NO 176 CUUUUCUUUUAGUUGCUGCUCUUUUSEQ ID NO 177 UUUUCUUUUAGUUGCUGCUCUUUUC SEQ ID NO 178UUUCUUUUAGUUGCUGCUCUUUUCC SEQ ID NO 179 UUCUUUUAGUUGCUGCUCUUUUCCASEQ ID NO 180 UCUUUUAGUUGCUGCUCUUUUCCAG SEQ ID NO 181CUUUUAGUUGCUGCUCUUUUCCAGG SEQ ID NO 182 UUUUAGUUGCUGCUCUUUUCCAGGUSEQ ID NO 183 UUUAGUUGCUGCUCUUUUCCAGGUU SEQ ID NO 184UUAGUUGCUGCUCUUUUCCAGGUUC SEQ ID NO 185 UAGUUGCUGCUCUUUUCCAGGUUCASEQ ID NO 186 AGUUGCUGCUCUUUUCCAGGUUCAA SEQ ID NO 187GUUGCUGCUCUUUUCCAGGUUCAAG SEQ ID NO 188 UUGCUGCUCUUUUCCAGGUUCAAGUSEQ ID NO 189 UGCUGCUCUUUUCCAGGUUCAAGUG SEQ ID NO 190GCUGCUCUUUUCCAGGUUCAAGUGG SEQ ID NO 191 CUGCUCUUUUCCAGGUUCAAGUGGGSEQ ID NO 192 UGCUCUUUUCCAGGUUCAAGUGGGA SEQ ID NO 193GCUCUUUUCCAGGUUCAAGUGGGAC SEQ ID NO 194 CUCUUUUCCAGGUUCAAGUGGGAUASEQ ID NO 195 UCUUUUCCAGGUUCAAGUGGGAUAC SEQ ID NO 196CUUUUCCAGGUUCAAGUGGGAUACU SEQ ID NO 197 UUUUCCAGGUUCAAGUGGGAUACUASEQ ID NO 198 UUUCCAGGUUCAAGUGGGAUACUAG SEQ ID NO 199UUCCAGGUUCAAGUGGGAUACUAGC SEQ ID NO 200 UCCAGGUUCAAGUGGGAUACUAGCASEQ ID NO 201 CCAGGUUCAAGUGGGAUACUAGCAA SEQ ID NO 202CAGGUUCAAGUGGGAUACUAGCAAU SEQ ID NO 203 AGGUUCAAGUGGGAUACUAGCAAUGSEQ ID NO 204 GGUUCAAGUGGGAUACUAGCAAUGU SEQ ID NO 205GUUCAAGUGGGAUACUAGCAAUGUU SEQ ID NO 206 UUCAAGUGGGAUACUAGCAAUGUUASEQ ID NO 207 UCAAGUGGGAUACUAGCAAUGUUAU SEQ ID NO 208CAAGUGGGAUACUAGCAAUGUUAUC SEQ ID NO 209 AAGUGGGAUACUAGCAAUGUUAUCUSEQ ID NO 210 AGUGGGAUACUAGCAAUGUUAUCUG SEQ ID NO 211GUGGGAUACUAGCAAUGUUAUCUGC SEQ ID NO 212 UGGGAUACUAGCAAUGUUAUCUGCUSEQ ID NO 213 GGGAUACUAGCAAUGUUAUCUGCUU SEQ ID NO 214GGAUACUAGCAAUGUUAUCUGCUUC SEQ ID NO 215 GAUACUAGCAAUGUUAUCUGCUUCCSEQ ID NO 216 AUACUAGCAAUGUUAUCUGCUUCCU SEQ ID NO 217UACUAGCAAUGUUAUCUGCUUCCUC SEQ ID NO 218 ACUAGCAAUGUUAUCUGCUUCCUCCSEQ ID NO 219 CUAGCAAUGUUAUCUGCUUCCUCCA SEQ ID NO 220UAGCAAUGUUAUCUGCUUCCUCCAA SEQ ID NO 221 AGCAAUGUUAUCUGCUUCCUCCAACSEQ ID NO 222 GCAAUGUUAUCUGCUUCCUCCAACC SEQ ID NO 223CAAUGUUAUCUGCUUCCUCCAACCA SEQ ID NO 224 AAUGUUAUCUGCUUCCUCCAACCAUSEQ ID NO 225 AUGUUAUCUGCUUCCUCCAACCAUA SEQ ID NO 226UGUUAUCUGCUUCCUCCAACCAUAA SEQ ID NO 227 GUUAUCUGCUUCCUCCAACCAUAAASEQ ID NO 228 GCUGCUCUUUUCCAGGUUC SEQ ID NO 229 UCUUUUCCAGGUUCAAGUGGSEQ ID NO 230 AGGUUCAAGUGGGAUACUA DMD Gene Exon 59 SEQ ID NO 231CAAUUUUUCCCACUCAGUAUU SEQ ID NO 232 UUGAAGUUCCUGGAGUCUU SEQ ID NO 233UCCUCAGGAGGCAGCUCUAAAU DMD Gene Exon 67 SEQ ID NO 234GCGCUGGUCACAAAAUCCUGUUGAAC SEQ ID NO 235 CACUUGCUUGAAAAGGUCUACAAAGGASEQ ID NO 236 GGUGAAUAACUUACAAAUUUGGAAGC

1-15. (canceled)
 16. An antisense oligonucleotide of 20 to 24nucleotides in length, wherein the antisense oligonucleotide comprisesat least 18 consecutive bases of a base sequence of the sequenceCUGUUGCCUCCGGUUCUGAAGGUG (SEQ ID NO: 115), in which uracil bases arethymine bases, wherein the antisense oligonucleotide is a morpholinophosphorodiamidate antisense oligonucleotide, and wherein the antisenseoligonucleotide induces exon 53 skipping of human dystrophin pre-mRNA.17. The antisense oligonucleotide of claim 16 that is 21 nucleotides inlength.
 18. A pharmaceutical composition, comprising the oligonucleotideof claim 16 and a pharmaceutically acceptable excipient.
 19. Thepharmaceutical composition, comprising the oligonucleotide of claim 17and a pharmaceutically acceptable excipient.
 20. A method for treatingDuchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD),comprising administering to a subject a therapeutically effective amountof the oligonucleotide of claim
 16. 21. A method for treating DuchenneMuscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD), comprisingadministering to a subject a therapeutically effective amount of theoligonucleotide of claim 17.